Gaurav R. Dwivedi

Drug Resistance Reversal Potential of Ursolic Acid Derivatives against Nalidixic Acid- and Multidrug-resistant Escherichia coli.

As a part of our drug discovery program, ursolic acid was chemically transformed into six semi‐synthetic derivatives, which were evaluated for their antibacterial and drug resistance reversal potential in combination with conventional antibiotic nalidixic acid against the nalidixic acid‐sensitive and nalidixic acid‐resistant strains of Escherichia coli. Although ursolic acid and its all semi‐synthetic derivatives did not show antibacterial activity of their own, but in combination, they significantly reduced the minimum inhibitory concentration of nalidixic acid up to eightfold. The 3‐O‐acetyl‐urs‐12‐en‐28‐isopropyl ester (UA‐4) and 3‐O‐acetyl‐urs‐12‐en‐28‐n‐butyl ester (UA‐5) derivatives of ursolic acid reduced the minimum inhibitory concentration of nalidixic acid by eightfold against nalidixic acid‐resistant and four and eightfold against nalidixic acid‐sensitive, respectively. The UA‐4 and UA‐5 were further evaluated for their synergy potential with another antibiotic tetracycline against the multidrug‐resistant clinical isolate of Escherichia coli‐KG4. The results showed that both these derivatives in combination with tetracycline reduced the cell viability in concentration‐dependent manner by significantly inhibiting efflux pump. This was further supported by the in silico binding affinity of UA‐4 and UA‐5 with efflux pump proteins. These ursolic acid derivatives may find their potential use as synergistic agents in the treatment of multidrug‐resistant Gram‐negative infections.

4‐Hydroxy‐α‐Tetralone and its Derivative as Drug Resistance Reversal Agents in Multi Drug Resistant Escherichia coli

The purpose of present investigation was to understand the drug resistance reversal mechanism of 4‐hydroxy‐α‐tetralone (1) isolated from Ammannia spp. along with its semi‐synthetic derivatives (1a–1e) using multidrug resistant Escherichia coli (MDREC). The test compounds did not show significant antibacterial activity of their own, but in combination, they reduced the minimum inhibitory concentration (MIC) of tetracycline (TET). In time kill assay, compound 1 and its derivative 1e in combination with TET reduced the cell viability in concentration dependent manner. Compounds 1 and 1e were also able to reduce the mutation prevention concentration of TET. Both compounds showed inhibition of ATP dependent efflux pumps. In real time polymerase chain reaction (RT‐PCR) study, compounds 1 and 1e alone and in combination with TET showed significant down expression of efflux pump gene (yojI) encoding multidrug ATP binding cassettes (ABC) transporter protein. Molecular mechanism was also supported by the in silico docking studies, which revealed significant binding affinity of compounds 1 and 1e with YojI. This study confirms that compound 1 and its derivative 1e are ABC efflux pump inhibitors which may be the basis for development of antibacterial combinations for the management of MDR infections from inexpensive natural product.

Antibacterial and Synergy of Clavine Alkaloid Lysergol and its Derivatives Against Nalidixic Acid‐Resistant Escherichia coli

Antibacterial activity of lysergol (1) and its semi‐synthetic derivatives (2–14) and their synergy with the conventional antibiotic nalidixic acid (NA) against nalidixic acid‐sensitive (NASEC) and nalidixic acid‐resistant (NAREC) strains of Escherichia coli were evaluated. Lysergol (1) and derivatives (2–14) did not possess antibacterial activity of their own, but in combination, they significantly reduced the minimum inhibitory concentration (MIC) of NA. All the derivatives showed two‐ to eightfold reduction in the MIC of NA against NAREC and NASEC. Further, lysergol (1) and its derivatives 10 and 11 brought down eightfold reductions in the MIC of tetracycline (TET) against multidrug‐resistant clinical isolate of E. coli (MDREC). Treatment of these strains with the combinations of antibiotics and lysergol and its derivatives 10 and 11 (at reduced concentrations) significantly decreased the viability of cells. In an another observation, lysergol and its derivatives 10 and 11 inhibited ATP‐dependent efflux pumps, which was evident by ATPase inhibition and down‐regulation of multidrug ABC transporter ATP‐binding protein (yojI) gene. These results may be of great help in antibacterial drug development from a very common, inexpensive, and non‐toxic natural product.

Phytol Derivatives as Drug Resistance Reversal Agents

Phytol was chemically transformed into fifteen semi‐synthetic derivatives, which were evaluated for their antibacterial and drug resistance reversal potential in combination with nalidixic acid against E. coli strains CA8000 and DH5α. The pivaloyl (4), 3,4,5‐trimethoxybenzoyl (9), 2,3‐dichlorobenzoyl (10), cinnamoyl (11), and aldehyde (14) derivatives of phytol ((2E,7R,11R)‐3,7,11,15‐tetramethyl‐2‐hexadecen‐1‐ol) were evaluated by using another antibiotic, tetracycline, against the MDREC‐KG4 clinical isolate of E. coli. Derivative 4 decreased the maximal inhibitory concentration (MIC) of the antibiotics by 16‐fold, while derivatives 9, 10, 11, and 14 reduced MIC values of the antibiotics up to eightfold against the E. coli strains. Derivatives 4, 9, 10, 11, and 14 inhibited the ATP‐dependent efflux pump; this was also supported by their in silico binding affinity and down‐regulation of the efflux pump gene yojI, which encodes the multidrug ATP‐binding cassette transporter protein. This study supports the possible use of phytol derivatives in the development of cost‐effective antibacterial combinations.

Preparative Isolation of Bioenhancer Loganetin from sf Alstonia scholaris by Fast Centrifugal Partition Chromatography

Fast centrifugal partition chromatography (FCPC) was successfully applied in the separation of close R f complex bioactive iridoid, loganetin directly from the ethyl acetate extract of Alstonia scholaris . The experiment was performed with a two-phase solvent system composed of methyl tert- butyl ether (MtBE)/ACN/Water (3:1.5:3 v/v/v) where the lower phase of the biphasic system, the aqueous layer containing 8 mM HCl, was the stationary phase, while the upper organic layer supplemented with 15 mM triethylamine TEA was designated as the mobile phase. From 1.5 g of EtOAc extract, 48 mg of loganetin (1) was obtained in 94.4% purity as determined by HPLC. The isolated compound (1) was characterized on the basis of its 1 H, 13 C–NMR, and ESI-MS spectroscopic data. Although loganetin does not possess antibacterial activity of its own, but in combination, it appreciably reduces the minimum inhibitory concentration (MIC) of nalidixic acid (NA) against nalidixic acid resistant (NAREC) and nalidixic acid sensitive (NASEC) strains of Escherichia coli . Loganetin, a very common, inexpensive, and non-toxic natural product may finds its application in the antibacterial drug development for treating multidrug-resistant Gram negative infections.

Antibiotics potentiating potential of catharanthine against superbug Pseudomonas aeruginosa

Multidrug resistance (MDR) put an alarming situation like preantibiotic era which compels us to invigorate the basic science of anti-infective chemotherapy. Hence, the drug resistant genes/proteins were explored as promising drug targets. Keeping this thing in mind, proteome of Pseudomonas aeruginosa PA01 was explored, which resulted in the identification of tripartite protein complexes (MexA, MexB, and OprM) as promising drug target for the screening of natural and synthetic inhibitors. The purpose of present investigation was to explore the drug resistance reversal potential mechanism of catharanthine isolated from the leaves of Catharanthus roseous. Hence, the test compound catharanthine was in silico screened using docking studies against the above receptors, which showed significant binding affinity with these receptors. In order to validate the in silico findings, in vitro evaluation of the test compound was also carried out. In combination, catharanthine reduced the minimum inhibitory concentration MIC of tetracycline (TET) and streptomycin up to 16 and 8 folds, respectively. Further, in time kill assay, catharanthine in combination with TET reduced the cell viability in concentration dependent manner and was also able to reduce the mutation prevention concentration of TET. It was also deduced that drug resistance reversal potential of catharanthine was due to inhibition of the efflux pumps.

Tricyclic Sesquiterpenes From Vetiveria zizanoides (L.) Nash as Antimycobacterial Agents

Two bioactive constituents, khusenic acid (1) and khusimol (2), were isolated and characterized from hexane fraction of Vetiveria zizanoides roots. Compounds, 1 and 2, were tested against the various drug‐resistant mutants of Mycobacterium smegmatis. The results showed that compound 1 was 4 times more active than the standard drugs ciprofloxacin (CF) and nalidixic acid (NA) against the ciprofloxacin (CSC 101) and lomefloxacin(LOMR5)‐resistant mutants, whereas the compound 2 was 2 times more active against the CSC 101 than the NA and CF. Further, these compounds were tested against the virulent strain H37Rv of Mycobacterium tuberculosis, which showed that 1 was two times more active than NA, while 2 was equally active to NA. In in silico docking study, 1 showed better binding affinity than 2 with both subunits of the bacterial DNA gyrase, which was further confirmed from the in vitro bacterial DNA gyrase inhibition study. The in silico ADME analysis of 1 and 2 showed better intestinal absorption, aqueous solubility and ability to penetrate blood–brain barrier. Finally, compound 2 was found safe at the highest dose of 2000 mg/kg body weight. Being edible, fragrant natural products, 1 and 2 will have advantage over the existing synthetic drugs.

Nano Particles: Emerging Warheads Against Bacterial Superbugs.

Infectious diseases are one of the major causes of morbidity and mortality in children in developing and underdeveloped countries. Limited knowledge of targets (cell wall synthesis, replication, transcription, protein synthesis) for antibiotics and lack of novel antibiotics have lead to an emergence of different level of resistance in bacterial pathogens. Multidrug resistance is the phenomenon by which the bacteria exerts resistance against the two or more structurally unrelated drugs/antibiotics. A common goal in the post-genomic era is to identify novel targets/drugs for various life threatening bacterial pathogens. Nanoparticles are broadly defined as submicron colloidal particles of size less than 1μm. Nanoparticles of size less than 100nm are the most promising warheads to overcome microbial drug resistance because they can act as antibacterial/antibiotic modulating agents at the site of infection and may have more than one mode of action. These nanoparticles will be of immense help in transporting drugs directly at the infected sites. Thus prevent drug resistance development to a great extent. In this review, the key mechanisms of resistance in bacterial superbugs have been discussed as well as how nanoparticles can overcome them. It is hypothesized that the nanoparticles can overcome the drug resistance via a novel mechanism of action. Additionaly, nanopaticles may also work synergistically with antibiotics via increased uptake, decreased efflux and inhibition of biofilm formation. The degradation by metallo beta lactamases and synthesis of porins may also be facilitated through these nanoparticles.

Synergy of clavine alkaloid ‘chanoclavine’ with tetracycline against multi-drug-resistant E. coli

The emergence of multi drug resistance (MDR) in Gram-negative bacteria (GNB) and lack of novel classes of antibacterial agents have raised an immediate need to identify antibacterial agents, which can reverse the phenomenon of MDR. The purpose of present study was to evaluate synergy potential and understanding the drug resistance reversal mechanism of chanoclavine isolated from Ipomoea muricata against the multi-drug-resistant clinical isolate of Escherichia coli (MDREC). Although chanoclavine did not show antibacterial activity of its own, but in combination, it could reduce the minimum inhibitory concentration (MIC) of tetracycline (TET) up to 16-folds. Chanoclavine was found to inhibit the efflux pumps which seem to be ATPase-dependent. In real-time expression analysis, chanoclavine showed down-regulation of different efflux pump genes and decreased the mutation prevention concentration of tetracycline. Further, in silico docking studies revealed significant binding affinity of chanoclavine with different proteins known to be involved in drug resistance. In in silico ADME/toxicity studies, chanoclavine was found safe with good intestinal absorption, aqueous solubility, medium blood–brain barrier (BBB), no CYP 2D6 inhibition, no hepatotoxicity, no skin irritancy, and non-mutagenic indicating towards drug likeliness of this molecule. Based on these observations, it is hypothesized that chanoclavine might be inhibiting the efflux of tetracycline from MDREC and thus enabling the more availability of tetracycline inside the cell for its action.

DbMDR: A Relational Database for Multidrug Resistance Genes as Potential Drug Targets

DbMDR is non‐redundant reference database of multidrug resistance (MDR) genes and their orthologs acting as potential drug targets. Drug resistance is a common phenomenon of pathogens, creating a serious problem of inactivation of drugs and antibiotics resulting in occurrence of diseases. Apart from other factors, the MDR genes present in pathogens are shown to be responsible for multidrug resistance. Much of the unorganized information on MDR genes is scattered across the literature and other web resources. Thus, consolidation of such knowledge about MDR genes into one database will make the drug discovery research more efficient. Mining of text for MDR genes has resulted into a large number of publications but in scattered and unorganized form. This information was compiled into a database, which enables a user not only to look at a particular MDR gene but also to find out putative homologs based on sequence similarity, conserved domains, and motifs in proteins encoded by MDR genes more efficiently. At present, DbMDR database contains 2843 MDR genes characterized experimentally as well as functionally annotated with cross‐referencing search support. The DbMDR database (http://203.190.147.116/dbmdr/) is a comprehensive resource for comparative study focused on MDR genes and metabolic pathway efflux pumps and intended to provide a platform for researchers for further research in drug resistance.